The present invention relates generally to methods and compositions involving linked stress proteins and human papillomavirus protein antigens for inducing an immune response against human papillomavirus protein antigens.
Infection with human papillomaviruses (HPV) is common, and the viruses can be transmitted sexually. It is estimated that between 20 and 80% of sexually active adults are infected. While a majority of infections are asymptomatic, infection can lead to development of genital warts and cancer of the anogenital tract. Genital warts have a prevalence of 1-5% among adults. About one percent of women worldwide are afflicted with cervical cancer, which is the most common cause of death in women under the age of 50. Cervical cancer is strongly associated with HPV, Frazer, Genitourin Med 72:398-403 (1996).
Presently, no effective therapeutic compositions or prophylactic compositions, i.e., vaccines, against HPV are available, and there is, therefore, a need for development of effective compositions. The prospects for a conventional killed or live attenuated vaccine appear to be poor. According to Frazer, HPV has not yet been propagated in cell culture, and the tumor-promoting effects of HPV infection as well as the complete species specificity of HPV represent additional difficulties that cannot be readily overcome (Frazer, Genitourin Med 72:398-403 (1996)). It has been proposed that the observation that major capsid protein, when expressed in eukaryotic cells, forms virus-like particles that are immunogenic without adjuvant may provide a basis for the development of a vaccine (Christensen et al., J Gen Virol 75:2271-6 (1994); see also PCT/EP95/03974 and PCT/US95/12914).
HPV belongs to the A genus of the papovaviridae family which also includes SV40 and polyomavirus. More than 68 different types of HPV have been characterized that are structurally highly related but are less than 50% identical at the DNA sequence level. All known types are epitheliotropic viruses that infect specific types of epithelium and frequently produce epithelial proliferations. Several types were identified in common warts. Twenty-three types are known to infect the female and male anogenital tracts. Anogenital diseases caused by these types of HPV range from Condylomata acuminata to invasive squamous cell carcinoma. HPV DNA can be identified in over 80% of women with biopsy-confirmed squamous intraepithelial lesions or cervical intraepithelial neoplasia. A few particular types, including HPV 16, 18, and 31, are associated strongly with high grade squamous intraepithelial lesions and invasive cancer of the cervix, vulva, penis and anus (Lorincz et al., Obstet Gynecol 79:328-37 (1992)). According to Frazer, cervical cancer is 90-95% associated with HPV. Frazer, Genitourin Med 72:398-403 (1996). HPV is not only associated with cancer of the anogenital system, but is also present in pharyngeal, laryngeal and bladder carcinomas (Brachman et al., Cancer Res 52:4832-6 (1992); Rotola et al., Int J Cancer 52:359-65 (1992)). A recent study reported that HPV DNA was also present in 30% of lung carcinomas tested. Types identified included HPV 6, 11, 16, 18, 31 and 33 (Soini et al., Thorax 51:887-893 (1996)). Hence, HPV types most often associated with cancer are 6, 11, 16, 18, 31 and 33, of which HPV 16 and 18, which are detected in more than 90% of cervical carcinomas (van Driel et al., Ann Med 28:471-477 (1996)), have been investigated most thoroughly.
Papillomaviruses are DNA viruses having a double-stranded, circular DNA genome of 7800 to 7900 base pairs, a nonenveloped virion and an icosahedral capsid made of 72 capsomers. The genome contains three major regions, one coding for late genes, one coding for early genes and a non-coding region (Park et al., Cancer 76:1902-1913 (1995)). The non-coding region is also referred to as upstream regulatory region. This region is about 400 base pairs long and contains an array of binding sites for the various transcription factors controlling expression of early and late genes. The late gene region has two separate open reading frames encoding viral capsid proteins L1 and L2. Protein L1 is the major capsid protein that is highly conserved among different HPV species. The early gene region includes six open reading frames, designated E1, E2, E4, E5, E6 and E7. Proteins E6 and E7 are oncoproteins critical for viral replication as well as for host cell immortalization and transformation. Proteins E1, E2 and E4 also play an important role in virus replication. In addition, E4 functions in the maturation of the virus. The role of E5 is less well known.
Cells from malignant tumors share two important growth characteristics. They are immortalized, i.e., they do no senesce, and they are capable of anchorage-independent growth. Introduction of HPV 16 or HPV 18 DNA into immortalized rodent cells results in their transformation, i.e., they acquire the ability to grow in the absence of substratum attachment and the capacity to form tumors when injected into mice (Crook et al., Proc. Natl. Acad. Sci. USA 85:8820-24 (1998)). A different result is obtained when HPV DNAs are introduced into early passage, non-immortalized cells: the cells become immortalized but are not transformed (Woodworth et al., Cancer Res. 48:4620-28 (1988)). Thus, one pathway by which tumors develop involves a change that results in immortalization of cells followed by expression of HPV genes that results in their transformation. The HPV genes involved in transformation of cells in vitro are those encoding E6 and/or E7 (Bedell et al., J Virol 61:3635-40 (1987)). Mechanisms by which the E6 and E7 proteins may cause cellular transformation have been proposed (Park et al., Cancer 76:1902-1913 (1995), and references cited therein).
E6 is a small (approximately 15,000 MW) polypeptide containing Zn-binding domains. A clue to its transforming function was provided by the observation that the protein binds p53. The p53 protein is a well known tumor suppressor protein that negatively regulates cell cycle progression and, consequently, cell growth and division. Binding of E6 to p53 results in the ubiquination and eventual degradation of the latter protein, which process involves another cellular protein termed xe2x80x9cE6-associated proteinxe2x80x9d. Consequently, cells expressing E6 will have a reduced basal level of p53. p53 levels are elevated in response to DNA damage. Such increased levels result in the enhanced expression of p21, an inhibitor of cyclin-dependent kinases, which protein mediates cell cycle arrest. This mechanism provides cells with a time window within which they can repair damaged DNA prior to its replication, which would result in the establishment of the damage/mutation. E6-mediated enhanced turnover of p53 may prevent the mechanism from operating. Recently, it was also found that E6 not only affects cell cycle regulation by virtue of accelerating degradation of p53, but also, more directly, by blocking p53 from interacting with DNA (Thomas et al., Oncogene 10:261-8 (1995)).
The E7 protein is a small (approximately 10,000 Mw), Zn-binding phosphoprotein capable of binding the retinoblastoma gene product Rb. Rb is a tumor suppressor binding to and inactivating transcription factor E2F. The latter factor controls transcription of a number of growth-related genes including those encoding thymidine kinase, c-myc, dihydrofolate reductase and DNA polymerase alpha. Rb-E2F complex formation prevents the expression of the latter genes in G0 and G1 phases, restricting their expression to the S phase where the Rb-E2F complexes are programmed to dissociate, liberating active transcription factor E2F. Formation of Rb-E7 complexes prevents formation of Rb-E2F complexes with the result of shortening pre-S phases, i.e., accelerating progression through the cell cycle. Correlative evidence for the importance of these mechanisms is provided by the observations that E6 proteins from highly oncogenic HPV types (e.g., HPV 16 and 18) have higher affinities for p53 than corresponding proteins from non-oncogenic types, and that E7 proteins from highly oncogenic types have higher affinities for Rb than corresponding proteins from non-oncogenic types.
In a majority of cervical cancers and precursor lesions. HPV DNA is integrated in the host cell genome (Cullen et al., J. Virol. 65:606-12 (1991)). It appears that in most cases, integration involves breakage of the HPV genomic DNA in the E1/E2 region, leaving the E6/E7 region intact. A consequence of the breakage in the E1/E2 region is an interruption of the open reading frame encoding two different E2 proteins, the smaller of which proteins functions as a transcriptional repressor of early gene expression. This leads to an upregulation of E6 and E7 expression.
The present invention relates to compositions for inducing an immune response against an HPV protein antigen in a subject to which they are administered. The immune response can be a humoral or cellular response, in particular a cell-mediated, cytolytic response to an HPV protein antigen. The compositions can be used prophylactically or therapeutically. In a prophylactic application, induction of an immune response refers to elicitation of immune reactions over a very low background of inherent immunity. In a therapeutic application, induction of an immune response in a subject refers to the generation of responses that exceed, either in magnitude or in quality, responses previously elicited by contact with HPV protein antigens exhibited either by the virus or by infected or transformed cells of the subject. In particular embodiments, the compositions are used to generate immune responses to tumor cells expressing and exhibiting an HPV protein antigen. In these embodiments, preferred HPV protein antigens targeted by the compositions are the E6 and E7 early viral proteins that are known to be consistently expressed in HPV-associated tumors. In one embodiment, the compositions comprise an HPV protein antigen joined to a stress protein (or heat shock protein (Hsp)). The HPV protein antigen may be joined to the stress protein by chemical conjugation, or antigen and stress protein may be joined at the nucleotide level permitting expression and isolation of a fusion protein containing both antigen and stress protein sequences. The compositions can be introduced into a subject or used ex vivo to stimulate and/or cause expansion of a subject""s immune cells targeting or mediating targeting of HPV or cells including tumor cells exhibiting an HPV protein antigen. The compositions are effective in stimulating an immune response when administered as nonparticulate (e.g., not as part of a virus or virus-like particle) proteinaceous solutions in the absence of adjuvant.
In another embodiment, the compositions comprise an expression vector including nucleic acid sequences encoding an HPV protein antigen and a stress protein. The expression vector further comprises sequence elements directing transcription and translation of the coding sequences and may also include elements facilitating delivery to and persistence or amplification of nucleic acids in cells of a subject. The expression vector can be introduced into cells of a subject, or it can be used to transduce a subject""s cells ex vivo, resulting in the expression of an HPV protein antigen-stress protein fusion protein that will induce an immune response against the HPV protein antigen.
The present invention also relates to compositions comprising an HPV protein antigen joined to a stress protein in combination with another pharmacologically acceptable component. In one embodiment, the composition comprises a conjugate comprising a stress protein joined with an HPV protein antigen. In another embodiment, the composition comprises a fusion protein (e.g., proteins expressed from pET65HE6 and pET65HE7) in which a stress protein is fused to an HPV protein antigen. The conjugates and fusion proteins of these compositions are also claimed as are expression vectors encoding and capable of directing the expression of a fusion protein comprising a stress protein and an HPV protein antigen sequence in a subject""s cells.
The present invention also relates to uses of the compositions to enhance immune responses against HPV protein antigens and, in particular embodiments, against tumors exhibiting an HPV protein antigen. The articles from the scientific literature and patent applications cited herein, especially those relating to the preparation and use of compositions of the invention, are incorporated by reference in their entirety.